Atherosclerotic cardiovascular disease (CVD), type 2 diabetes (T2D) and metabolic syndrome (MetS) exert a huge public burden that will accelerate with the growing rates of obesity. These conditions share a pro- inflammatory state, yet shared underlying mechanisms remain unclear. The community of commensal microbes that reside in the gut has emerged as a modulator of inflammation, immune function and metabolism. Genetic knockout of innate immune components can recapitulate many aspects of MetS that are mediated by altered microbiota. Studies in human disease cohorts show the feasibility of finding microbial profiles associated with CVD, T2D and MetS, and implicate specific pathways of microbial metabolism (e.g., biosynthesis of short-chain fatty acids [SCFA] and trimethylamine-N-oxide [TMAO]) that interact with diet and may modulate inflammation, metabolism, and risk of CVD, MetS and T2D. However, our understanding of how these pathways interact with microbial taxonomy and function, host pathways and environment (e.g., diet) remains incomplete. The overall hypothesis of this proposal is that the function of the gut microbiota may provide a unifying mechanism for many of the overlapping metabolic and inflammatory phenotypes observed in CVD, MetS and T2D. Specifically, we will test focused hypotheses about the roles of the SCFA and TMAO pathways, including their relation to microbial species and function, diet, host genotype, and CVD, MetS and T2D. We will also pursue secondary, unbiased analyses into the association of microbiota composition and function with CVD, MetS and T2D. We will test these hypotheses in the Framingham Heart Study (FHS) Gen3 and Omni2 cohorts, a middle-aged, multi-ethnic, community-based sample of 3800 men and women. In Aim 1, we will use 16S rRNA gene sequence and calculated microbial pathway/functional information to test hypotheses about the relation of stool SCFA and plasma TMAO levels to gut microbial species and function, diet, and prevalent CVD, MetS and T2D. In Aim 2, we will identify host genes that influence: (a) stool SCFA and plasma TMAO levels, (b) microbial taxa/functions associated with stool SCFA or plasma TMAO, and (c) taxa/functions associated with prevalent CVD, MetS or T2D (while identifying host features shared vs. private to more than one of these diseases). In Aim 3, we will test whether meta-genomic, metatranscriptomic and metabolomics microbiota profiles of stool samples at baseline and in close proximity to incident CVD or MetS confer additional mechanistic insights. Understanding how specific microbial pathways relate to diet, host genes, and disease risk may ultimately lead to novel therapies that modulate microbial function or host- microbe functional interactions. This application combines FHS genotypes, phenotypes and expertise in CVD and metabolic disease with a team experienced in the Human Microbiome Project and drawing biomedical insights from microbiome studies. The data from this study will be a community resource and benefit microbiota studies in the broader community.